The uptake of low-density lipoprotein (LDL) and the accumulation of cholesterol were assessed in opossum kidney (OK) and Madin-Darby canine kidney (MDCK) cells. OK and MDCK cells were grown to confluency on Millicell well inserts. The uptake of human LDL across the apical and basolateral surfaces of OK and MDCK cells was assessed by the degradation of internalized (125I)LDL to trichloroacetic acid-soluble products. LDL uptake via the apical surface of OK cells increased linearly with LDL concentration, indicating nonreceptor-mediated uptake. In contrast, LDL uptake via the basolateral surface of OK cells and both apical and basolateral surfaces of MDCK cells followed a saturable pattern. In addition, (125I)LDL bound to the apical membrane of MDCK cells, but not to the apical membrane of OK cells, was displaced by heparin and by excess of unlabeled LDL. Exposure to LDL (100 mg/mL) resulted in an increase in total cholesterol content of OK and MDCK cells (23 and 18%, respectively). Most of the increase in total cholesterol content with LDL exposure resulted from increased free cholesterol content in MDCK cells and esterified cholesterol in OK cells. The differences in cholesteryl ester formation were consistent with the slower rates of (14C) oleate incorporation into cholesteryl ester and lower cholesterol esterifying activity observed in MDCK cells compared with that in OK cells. These results demonstrate that LDL uptake can be receptor or nonreceptor mediated, depending upon the renal cell type and the surface exposed to LDL, and that LDL exposure leads to increased cholesterol content in OK and MDCK cells.